viruses Article The Alphaviral Capsid Protein Inhibits IRAK1-Dependent TLR Signaling V. Douglas Landers 1,2 , Daniel W. Wilkey 3, Michael L. Merchant 3, Thomas C. Mitchell 1 and Kevin J. Sokoloski 1,2,* 1 Department of Microbiology and Immunology, School of Medicine, University of Louisville, Louisville, KY 40202, USA; [email protected] (V.D.L.); [email protected] (T.C.M.) 2 Center for Predictive Medicine and Emerging Infectious Diseases, University of Louisville, Louisville, KY 40202, USA 3 Department of Medicine, Division of Nephrology and Hypertension, School of Medicine, University of Louisville, Louisville, KY 40202, USA; [email protected] (D.W.W.); [email protected] (M.L.M.) * Correspondence: [email protected] Abstract: Alphaviruses are arthropod-borne RNA viruses which can cause either mild to severe febrile arthritis which may persist for months, or encephalitis which can lead to death or lifelong cognitive impairments. The non-assembly molecular role(s), functions, and protein–protein inter- actions of the alphavirus capsid proteins have been largely overlooked. Here we detail the use of a BioID2 biotin ligase system to identify the protein–protein interactions of the Sindbis virus capsid protein. These efforts led to the discovery of a series of novel host–pathogen interactions, including the identification of an interaction between the alphaviral capsid protein and the host IRAK1 protein. Importantly, this capsid–IRAK1 interaction is conserved across multiple alphavirus species, including arthritogenic alphaviruses SINV, Ross River virus, and Chikungunya virus; and encephalitic alphaviruses Eastern Equine Encephalitis virus, and Venezuelan Equine Encephalitis Citation: Landers, V.D.; Wilkey, virus. The impact of the capsid–IRAK1 interaction was evaluated using a robust set of cellular model D.W.; Merchant, M.L.; Mitchell, T.C.; systems, leading to the realization that the alphaviral capsid protein specifically inhibits IRAK1- Sokoloski, K.J. The Alphaviral Capsid Protein Inhibits IRAK1-Dependent dependent signaling. This inhibition represents a means by which alphaviruses may evade innate TLR Signaling. Viruses 2021, 13, 377. immune detection and activation prior to viral gene expression. Altogether, these data identify novel https://doi.org/10.3390/v13030377 capsid protein–protein interactions, establish the capsid–IRAK1 interaction as a common alphavirus host–pathogen interface, and delineate the molecular consequences of the capsid–IRAK1 interaction Academic Editor: Karla Helbig on IRAK1-dependent signaling. Received: 3 February 2021 Keywords: alphavirus; capsid; IRAK1; toll like receptors (TLR) Accepted: 26 February 2021 Published: 27 February 2021 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in Alphaviruses are positive-sense RNA viruses which are primarily spread via vector- published maps and institutional affil- competent mosquito species [1]. Collectively, and often on a seasonal basis, the members iations. of genus Alphavirus are responsible for local, regional, and global outbreaks of clinically severe illness [2–14]. Alphaviruses may be broadly classified via their predominant symp- tomology as either arthritogenic or encephalitic. Arthritogenic alphaviruses, such as Sindbis virus (SINV; the prototypic alphavirus), Ross River virus (RRV), Semliki Forest Copyright: © 2021 by the authors. virus (SFV), and Chikungunya virus (CHIKV), exhibit low mortality despite often causing Licensee MDPI, Basel, Switzerland. febrile illness with debilitating multifocal arthritis [15–23]. In some instances, the multi- This article is an open access article focal arthritis may persist for several months to years past the resolution of the primary distributed under the terms and infection [15–17,24–26]. In contrast to arthritogenic alphaviruses, encephalitic alphaviruses conditions of the Creative Commons Attribution (CC BY) license (https:// can exhibit significant mortality and life-altering neurological sequelae, primarily in young creativecommons.org/licenses/by/ children and adolescents [27–30]. Despite the clear impact of alphaviruses on global human 4.0/). health and quality of life in developing and developed communities alike, there are no Viruses 2021, 13, 377. https://doi.org/10.3390/v13030377 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 377 2 of 27 clinically proven antiviral therapeutics, or safe and effective vaccines to mitigate the public health threat posed by alphaviruses. An infectious alphavirus particle is relatively simple in design. Measuring approxi- mately 70 nm in diameter, an alphavirus particle features an RNA cargo surrounded by two concentric icosahedral protein layers divided by a host-derived lipid envelope [31,32]. The viral glycoproteins E1 and E2 (and in some instances E3) are ordered in an icosahedral array projecting from the external surface of the viral envelope [31,32]. Several copies of the viral 6K and TF proteins, precisely how many is unknown at this point, are associated with the viral envelope [33,34]. The C-terminal endodomain of the E2 protein interacts with the viral capsid (CP) protein, which also forms an icosahedral structure which is symmetrically aligned with the viral glycoprotein spikes. The CP protein is the sole viral protein component of the nucleocapsid core and is the most abundant viral protein in the mature viral particle [1]. The alphaviral entry pathway initiates with the interaction of the host receptor with the viral E2 glycoprotein, resulting in the endocytosis of the viral particle, and culminates with the delivery of the nucleocapsid core to the host cytoplasm [1]. The nucleocapsid core then rapidly disassembles, releasing the CP protein from the viral genomic RNA, the latter of which interacts with host factors to engage the translational machinery to initiate the synthesis of the viral replicase complex. While still becoming better understood at the molecular level, the fate of genomic RNA is straightforward. In contrast, despite being the predominant viral component released to the host cytoplasm, the role of the viral CP protein after entry is less understood. Prior work by the Sokoloski lab identified a series of non-assembly CP–RNA (naCP–RNA) interactions which functioned during the early stages of viral infection [35]. The disruption of the naCP–RNA interactions negatively impacted viral particle infectivity, which correlated with decreased viral RNA stability in cellular models of infection [35]. Collectively, these data led us to hypothesize that the alphaviral CP proteins which are delivered as part of the nucleocapsid core may function to influence the host cell environment after disassembly. We further postulated that the molecular activities of the CP protein are dependent on the formation of host–pathogen protein–protein interactions which impart new functionality to the CP protein complex, or disrupt the activities of the normal cellular protein complexes. The above overarching hypotheses, and the absence of a comprehensive analysis of the alphavirus CP protein–protein interaction data in the knowledgebase, led us to examine the protein–protein interactions of the SINV CP protein using an innovative approach. Here we detail the use of an adapted BioID approach to identify the putative host–pathogen interactions of the SINV CP protein [36,37]. This discovery approach led to the identification and validation of a novel alphaviral host–pathogen interaction—the interaction of the alphaviral CP protein with the host IRAK1 protein. The host IRAK1 protein is a critical component of the TLR and IL-1R signal transduction pathways, and thus the CP–IRAK1 interaction may negatively impact the detection and response to TLR and IL1R ligand binding [38,39]. Using a robust series of state-of-the-art model systems, we assessed the impact of the CP–IRAK1 on IRAK1-dependent signaling and found that that the alphaviral CP protein was capable of significantly inhibiting IRAK1-dependent TLR signaling. Importantly, the SINV CP proteins delivered from viral particles during viral entry were sufficient to mask TLR agonist detection, regardless of viral particle infectivity. Taken together, the data presented in this study significantly contribute to the field by i) using an unbiased approach to identify putative CP–protein interactions, and ii) delineating a novel mechanism by which the host innate immune response is evaded during the earliest stages of alphaviral infection prior to viral gene expression. 2. Materials and Methods 2.1. Tissue Culture Cells HEK293 (ATCC CRL-1573) and BHK-21 (ATCC CCL-10) cells were cultured in Mini- mal Essential Media (MEM; Cellgro Mediatech, Inc, Manassas, VA USA), supplemented with 10% Fetal Bovine Serum (FBS; Corning, Corning, NY USA), 1× Penicillin/Steptomycin Viruses 2021, 13, 377 3 of 27 (Pen/Strep; Corning, Corning, NY USA), 1× Non-Essential Amino Acids (NEAA; Corn- ing, Corning, NY USA), and L-glutamine (Corning, Corning, NY USA). HEK293-derived reporter cells, namely HEK-Blue hTLR3, HEK-Blue hTLR4, and HEK-Blue hTLR7 (Invivo- gen, San Diego, CA USA), were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Corning, Corning, NY USA) supplemented with 4.5 g/L glucose, 10% FBS, 1× Pen/Strep, and 1× Normocin (Invivogen, San Diego, CA USA). To maintain genetic homogeneity, the HEK-Blue tissue culture cells were maintained at low passage number and supplemented with the appropriate selection antibiotics on alternating passages